The cysteinyl leukotrienes (CysLTs) mimic many of the features of asthma and are implicated in its pathophysiology. Little, however, is known about the effects of the CysLTs on airways remodeling. In this study the effects of leukotriene D4 (LTD4) on human airway smooth muscle (HASM) cell proliferation and expression of extracellular matrix proteins were investigated. LTD4 (0.1-10 microM) alone had no effect on DNA synthesis in HASM. LTD4, however, markedly augmented proliferation induced by the mitogen, epidermal growth factor (EGF, 1 ng/ml). The potentiating effect of LTD4 (1 microM) on EGF-induced DNA synthesis was abolished by pranlukast (1 microM) or pobilukast (30 microM), but unaffected by zafirlukast (1 microM). In contrast, pranlukast (pKB = 6.9), pobilukast (pKB = 7.0), and zafirlukast (pKB = 6.5) had equivalent potencies for inhibition of LTD4-induced contraction in human bronchus. LTD4 (0.1 or 10 microM) did not increase the total messenger RNA expression of the extracellular matrix proteins (pro-alpha[I] type I or alpha1[IV] type IV collagen), elastin, biglycan, decorin, and fibronectin, and did not influence tumor growth factor-beta (10 ng/ml)-induced effects on the expression of these proteins in HASM cells. These data indicate that LTD4 augments growth factor-induced HASM proliferation but does not alter the expression of various extracellular matrix components. The observed differences in sensitivity to the antagonists suggests that the former phenomenon may be mediated by a CysLT receptor distinct from that which mediates LTD4-induced HASM contraction. Collectively, these results provide preliminary evidence that CysLTs may play a role in airways remodeling in asthma.

Regulation of phosphatidylinositol (PI) 3-kinase plays an important role in modulating cellular function. We have previously shown that transforming growth factor (TGF)-β1 inhibited epidermal growth factor (EGF)-induced human airway smooth muscle (hASM) cell proliferation and that PI 3-kinase activation is a necessary signaling event in mitogen-induced hASM cell growth. In this study, we postulated that TGF-β1 may modulate EGF-induced PI 3-kinase activation. To date, no study has examined the effects of TGF-β1 on PI 3-kinase activity. In cultured hASM cells, EGF induced a 5.7 ± 1.2-fold activation of PI 3-kinase compared with diluent-treated cells. Although TGF-β1 alone did not alter PI 3-kinase activation, TGF-β1 markedly enhanced EGF-induced PI 3-kinase activity, with a 16.6 ± 1.9-fold increase over control cells treated with diluent alone. EGF significantly increased the association of PI 3-kinase with tyrosine phosphorylated proteins, and TGF-β1 pretreatment before EGF stimulation apparently did not alter this association. Interestingly, TGF-β1 did not modulate EGF-induced p70 S6 kinase activity, which is important for the progression of cells from the G0 to the G1 phase of the cell cycle. Immunoprecipitation of type I and type II TGF-β receptors showed that PI 3-kinase was associated with both type I and type II TGF-β receptors. TGF-β1, however, enhanced PI 3-kinase activity associated with the type I TGF-β receptor. Although in some cell types inhibition of PI 3-kinase and treatment of cells with TGF-β1 mediate apoptosis, cell cycle analysis and DNA ladder studies show that PI 3-kinase inhibition or stimulation of hASM cells with TGF-β1 did not induce myocyte apoptosis. Although the inhibitory effects of TGF-β1 on hASM cell growth are not mediated at the level of PI 3-kinase and p70 S6 kinase, we now show that activation of the TGF-β1 receptor modulates PI 3-kinase activity stimulated by growth factors in hASM cells.

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